CaMKII is a leading candidate as a synaptic memory molecule. It is localized at synapses, persistently activated after LTP induction and can itself stimulate reactions that strengthen the synapse. Importantly, mutations in the kinase that block persistent activation produce a strong reduction of LTP and major deficits in memory. This proposal focuses on what happens to CaMKII after it is activated and seeks to critically test whether the kinase indeed serves as a synaptic memory molecule. Our recent work shows that LTP induction causes a persistent translocation of the kinase to the synapse that lasts for at least an hour. Our overall goal is to determine whether these persistent changes in the kinase are involved in the persistent changes of the synapse. In the first Aim, we will test more generally whether the CaMKII content of synapses is related to the strength of the synapse. We will examine whether translocation extends to late LTP, whether depotentiation and LTD produce a reduction in synaptic CaMKII content and whether, under basal conditions, there is a correlation between the CaMKII content of individual synapses and their strength, as measured with 2-photon glutamate uncaging. A second Aim concerns the targets of translocation. We will use EM method to determine whether the target of translocation is the postsynaptic density itself. In other experiments, cells will be transfected with agents that interfere with the binding of CaMKII to specific molecular targets at synapses. These experiments may shed light on which CaMKII binding partners are important for translocation and LTP. In a final set of experiments we seek to understand the functional role of long-term changes in the kinase and specifically whether they have a role in maintaining synaptic memory. We first will test whether the kinase that translocates to the PSD is persistently phosphorylated. Furthermore, we will test whether the phosphorylated kinase has to phosphorylate other proteins to produce LTP or whether it can promote LTP by acting as a structural protein. The findings obtained are relevant to the interpretation of the next experiments, in which kinase inhibitor is used to test the functional role of CaMKII. In these experiments, we will be able to directly measure the effect of inhibitor on the phosphorylation state of PSD CaMKII and determine whether potentiation declines as the kinase becomes dephosphorylated. These experiments will provide strong tests of the biochemical mechanisms that underlie synaptic memory. This work has direct relevance to the search for the deficits that underlie diseases of memory and other health problems, such as chronic pain, addiction and epilepsy, where abnormalities in the strength of synapses have been implicated.